Vessel for accurate analysis

ABSTRACT

Vessels used to contain reaction mixtures and that allow for accurate optical or visual detection are described. Vessels that are an integrated tube- and cap assembly connected via one or more hinge straps or devices to other vessels or caps and/or tubes and caps with different light transmitting properties are also described.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to vessels used to containreaction mixtures and allowing for accurate optical or visual detection.More specifically the invention relates to a family of those vesselsknown as tube strips with attached caps and single tubes with attachedcaps.

2. Description of Related Art

Small amounts of reaction mixtures are commonly stored in disposableplastic tubes, which are closed with caps. There are a number ofcommercially available vessels of this type and accompanying sealingsystems, such as microcentrifuge tubes and tubes used, for example,during heating, chilling or thermal cycling (e.g. from ABgene, Applera,Axygen and BIOplastics). The vessels are available in a number offormats including single tubes, tubes arranged in tray type arrays,typically known as multi-well plates, and also strips of attached tubes,typically arranged with a tube center-to-center distance matching thatfound in one dimension of multi-well plates. The single tubes and stripsof tubes are generally sealed by means of a molded cap or strip of capsthat fit securely into the mouth(s) of the tube(s). These caps may beseparate or integral to the tube(s). It is reasonable to say that thesingle tubes and strips of tubes are produced exclusively via typicalhigh-pressure injection molding processes.

Because of the good qualities obtained during the manufacture of vesselsusing high-pressure injection molding, these vessels are suited forapplications using optical detection, such as thermal cycling.

The products of a polymerase chain reaction (PCR) are typically analyzedusing end point analysis techniques such as gel electrophoresis. Thesetechniques lack speed and accuracy and are useful primarily to determinerelative quantities of known and unknown samples and are best used as asimple measure of whether target sequences are present or not.

A next generation family of instruments now exists which enable the userto monitor a PCR process in real time by utilizing protocols involvinglight excitation and detection. These instruments are generally known asqPCR (quantitative PCR) instruments. While there are many protocols,which may be performed in these instruments, two common ones are knownas the SYBR green method and the fluorescent reporter probe method. Inthe SYBR green method, a DNA binding dye binds to all newly synthesizeddouble-stranded DNA and an increase in fluorescence intensity ismeasured allowing initial concentrations to be determined. In thefluorescent reporter probe method only a probe sequence is quantifiedand not all double stranded DNA. It is commonly carried out with afluorescent reporter and a quencher held in adjacent positions. Upon thebreaking of the probe fluorescence may be detected, since more and moreof the fluorescent reporter is liberated from its quencher, resulting inan easily detectable increase in fluorescence.

It may be noted that both of these protocols, as well as allconventional qPCR protocols, typically rely upon the introduction ofexcitation light into the sample inside the vessel by way of passing itthrough the vessel-sealing device. In the case of strip tubes and singletubes the sealing device is typically a cap. It may further be notedthat detection of the emitted signal relies upon the fluorescence beingdetected through that same sealing device. It may also be noted that, inthe instance where many tubes are in close proximity to one another inthe thermal cycling device and a light detection protocol is employed,tube opacity is desirable to contain the light signal within theindividual tubes thereby increasing detection accuracy. A white tube isparticularly advantageous.

The type of vessels generally used in applications such as thermalcycling is available in three different formats:

1. Strips of tubes with attached optical caps, typically strips ofeither 8 or 12 tubes:

-   -   In the prior art these are always injection molded of one        polymer, optionally containing pigment or dye, making the        complete assembly either clear or opaque, generally clear. While        the product has the benefit of being easy to handle because of        the attached cap, this product is less than optimal because one        may not procure it for example in an opaque tube/clear cap        configuration or a reflective tube/clear cap configuration or a        configuration, wherein the tube and the cap both are colored,        but with different colors. In some known vessels, the surface of        the tube is “frosted”, but this does not provide a completely        opaque surface.

2. Single tubes with attached caps:

-   -   In the prior art this form of product is not optimal for the        reasons cited above.

3. Strips of tubes without caps:

-   -   Strip tubes without caps are available also in white color. In        this format the sealing device is a separate strip of caps made        of a clear polymer. While this combination of products will work        for applications using optical detection, it lacks the economy        and ease of use of the formats with integrated optical caps.

As a general example of available vessel formats, US 20050084957presents a tube and a method for achieving accurate temperature controlfor a large number of samples arranged in a microtiter plate formatduring very rapid thermal cycling PCR protocols.

SUMMARY OF THE INVENTION

It is an aim of the present invention to provide a novel integratedtube-and-cap assembly, wherein the tube and the cap have differentoptical properties.

Particularly, it is an aim of the present invention to provide a novelvessel for accurate optical detection.

These and other objects, together with the advantages thereof over knownvessels and methods, are achieved by the present invention, ashereinafter described and claimed.

The present invention concerns a vessel comprising an integratedtube-and-cap assembly, wherein one or more tube is connected by way of ahinge strap or device to one or more cap.

More specifically, the vessel of the present invention is characterizedby what is stated in the characterizing part of claim 1.

Further, the use of the present invention is characterized by what isstated in claims 16 and 17 and the method of the present invention ischaracterized by what is stated in the characterizing part of claim 18.

Considerable advantages are obtained by means of the invention. Thus,the present invention provides a unique single combined tube and capstructure composed of two separate materials, said combined structurehaving the economy, utility and ease of use of prior art single materialproducts combined and which may also embody the features of preventionof tube-to-tube light transmission and internal light reflection,thereby improving signal quality

Next, the invention will be described more closely with reference to theattached drawing and a detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a perspective view of the tube-and-cap assembly of thepresent invention. In FIG. 1 a the assembly of one of the embodiments ofthe invention is shown. FIG. 1 b is a close-up of the tube portion ofthe assembly and FIG. 1 c is a close-up of the cap portion.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns a vessel comprising an integratedtube-and-cap assembly, wherein one or more tube(s) is connected by wayof one or more hinge strap(s) or device(s) to one or more cap(s).

Particularly, the invention concerns a vessel comprising an integratedtube-and-cap assembly, which assembly has been fabricated in one pieceand wherein the tube and the cap have different light transmittingproperties, since the material that either one of the tube and the capis fabricated from comprises a colorant, which manipulates its lighttransmitting properties, while the other one is fabricated from amaterial, which lacks such colorant.

The vessel can comprise a single tube or a one-dimensional array havinga plurality of sample tubes arranged in a line (microtiter “strip”)(FIG. 1 a).

The vessel comprises an assembly, wherein (as in FIG. 1) a tube portion(FIG. 1 b) comprises the following parts:

-   -   1 an upper surface    -   2 an opening    -   3 an upper wall portion    -   4 a lower wall portion    -   5 a bottom portion    -   6 a neck        connected (FIG. 1 c) by way of:    -   7 a hinge strap or device        to a cap portion (FIG. 1 c) comprising the following parts:    -   8 a cylindrically shaped portion    -   9 a closed dome-shaped or flat portion    -   10 a circumferential shoulder

The “tube” or particularly the “strip” of tubes may comprise an uppersurface 1, from which the actual sample tube part protrudes downwardly,while its opening 2 remains on the upper surface 1. The tube is formedof an upper wall portion 3, which preferably has a cylindrical shape, alower wall portion 4, which may also have a cylindrical shape or it mayhave a conically beveled shape, preferably a conically beveled shape,and a bottom portion 5.

The lower wall portion 4 extends from the upper wall portion 3continuously, preferably so that the wall thickness is reduced as thedistance from the upper wall portion 3 increases. Thus, the lower wallportion 4 is connected to the upper wall portion 3 at one end, which inthe conically beveled alternative shape is the wider end, and to thebottom portion 5 at the other end. The bottom 5 of the tube thus closesthe structure. The bottom portion 5 can be made thicker than the lowerwall portion 4 to increase the strength of the structure but can alsohave the same thickness as the lower wall portion 4. If the vesselcomprises a strip of tubes, the tubes are typically connected toneighboring tubes from the upper wall portions 3 by necks 6. In thatcase, each of the sample tubes in the vessel is connected from itscylindrical upper wall portion 3 to neighboring tubes by a neck 6. Thus,the necks 6 form the upper surface 1 of the strip. The sample tubes canalso comprise shoulders, as described in the published patentapplication no. US 2005/0,084,957.

The tube is connected to a cap by way of a “hinge strap or device” 7,herein also called a “hinge-like structure”.

The term “cap” is used herein to describe any designs capable of sealinga sample tube, thereby preventing evaporation of the sample before orduring storage, centrifuging, thermal cycling or any other process thetube, the cap and the sample may be used for. The cap can also bedescribed for example as a stopper, a closure or a plug, and it ispreferably manufactured from a polymeric material, such as a plastic.The cap is preferably pivotally attached to the cylindrical part of thetube and includes an essentially cylindrically shaped portion 8 that isremovably insertable into said sample tube and is dimensioned to give atight fit, thereby preventing evaporation of the tube contents. The capfurther comprises a resiliently deformable closed dome-shaped or flatportion 9, which closes the structure. The dome-shaped or flat portion 9extends away from the cylindrically shaped portion 8. The cap furthercomprises a circumferential shoulder 10, attached to and interposedbetween the mentioned cylindrically shaped portion 8 and the dome-shapedor flat portion 9, extending radially outwards from the mentionedportions 8,9.

The “light transmitting properties” of the materials used in thefabrication of the assembly of the present invention are mainly dividedinto four groups, i.e. opaque, optically clear, colored and reflective.These properties may be altered/manipulated for example by adding one ormore colorants to the material that the tube, the cap or the hinge strapis fabricated from during the fabrication (not shown in FIG. 1).

The term “colorant”, as used herein, is meant to include pigments,nanoparticles and any other agents, which alter the light transmittingproperties of the base material of the tube, for example thethermoplastic material.

The term “opaque”, as used herein to describe a material, means that thematerial, such as the wall of the tube, blocks or reflects light. Theterm “essentially opaque”, as used herein, means that the mentionedmaterial has at least a portion, which is completely opaque, i.e.transmits very little light by reflecting or blocking most of it. Theopaque portion may be a portion of the tube wall facing the directionsof adjacent tubes, or other possible sources of an emitted signal, andmay cover for example the cylindrical portion 3 of the tube. This is dueto the cylindrical portion 3, at least in some cases, reaching above theupper edge of the well of the sample holder that the tube has beenplaced in. The walls of the wells prevent tube-to-tube lighttransmission, whereby only reflections from the walls of the wells(generally made of metal) may affect the accuracy of the results in thelower parts of the tube.

The term “optically clear”, as used herein to describe a material, meansthat the material, such as the cap, transmits light (being transparentor translucent). The term “essentially optically clear”, as used herein,means that the mentioned material contains at least a portion, which isoptically clear, i.e. transmits light. The optically clear portion maybe a portion of the cap covering at least a surface large enough for anemitted signal to pass, i.e. the portion may consist of only an opticwindow. The optic window may, for example, cover only the dome-shaped orflat portion 9 of the cap. For the achievement of a reliable detection,the clear material is required to be homogenous.

The term “homogenous”, as used herein, is meant to describe the materialthat the vessel of the present invention is manufactured of, whichmaterial has a homogenous surface with a homogenously distributed color.

“Thermal cyclers” are instruments commonly used in molecular biology forapplications such as the polymerase chain reaction (PCR) and cyclesequencing, and a wide range of instruments are commercially available.“qPCR” is a quantitative polymerase chain reaction, i.e. a modificationof the polymerase chain reaction, which is used to rapidly measure thequantity of a product of the polymerase chain reaction. It is preferablydone in real-time.

According to one embodiment of the present invention, the tube portionof the tube-and-cap assembly has altered light transmitting properties,making it opaque, colored or reflective. According to this embodiment,the cap portion has unaltered light transmitting properties, making itcolored, but of a different color than the tube portion, or opticallyclear.

According to another embodiment of the present invention, the capportion of the assembly has altered light transmitting properties, whilethese properties are unaltered in the tube.

These embodiments, as described herein, rely on the base material usedin the fabrication of the assembly being optically clear or colored. Thepresent invention, however, is also meant to include base materials thatare opaque, frosted or reflective, whereby altering the lighttransmitting properties may make the material optically clear inaddition to the alterations described above. The assembly of the presentinvention may thus comprise an opaque tube and a clear cap, a clear tubeand an opaque cap, a reflective tube and a clear cap, a clear tube and areflective cap, a tube and a cap of different colors, a frosted tube anda colored cap, a colored tube and a frosted cap, a frosted tube and anopaque cap, an opaque tube and a frosted cap, a reflective tube and afrosted cap as well as a frosted tube and a reflective cap.

According to a preferred embodiment of the present invention, the tubeis essentially opaque and is connected by way of a hinge strap or device7 to an essentially optically clear cap. Preferably, the essentiallyopaque part of the tube is of a uniform, homogenous, opaque color, suchas black or white, giving a complete opacity and thus a uniform lightdetection. More preferably, the opaque part of the tube is white.

According to a particularly preferred embodiment, the cap is fabricatedcompletely from an optically clear material. The optically clearmaterial is chosen from materials allowing for the introduction ofexcitation light and for the detection of an optical signal.

According to another particularly preferred embodiment, the tube isfabricated completely from an opaque material. Manufacturing not onlythe upper portion but also the lower portion of the tube from an opaquematerial prevents not only tube-to-tube light transmission, but alsosuccessfully prevents reflection of light from the walls of the wells ofa laboratory instrument, such as a thermal cycling device or othersimilar device, from entering the tube. Further, a frosted tube, as theone used by BIOplastics, partly prevents light from entering orre-entering the tube through the wall of the tube, but an opaque tube,that according to the definition above blocks or reflects light, givesan even more accurate and more repeatable result, since the surface ofthe opaque tube provides a more complete prevention of lighttransmission.

According to a further preferred embodiment, the tube portion isfabricated from an optically clear material, whereas the cap portion isfabricated from an opaque material.

The base material of the sample tube and the cap preferably comprises athermoplastic material, which will withstand the conditions typical for,e.g. thermal processing of biological samples, involving heating cyclesincreasing the temperature up to more than 80° C. In addition, thematerial should exhibit good hydrophobicity and low interference withmolecular biological reactions. Examples of suitable materials includevarious polyolefin grades, polyesters and polycarbonates. A particularlypreferred material is polypropylene, preferably of a grade suitable formelt processing, e.g. by injection molding, pressure forming, vacuumforming, extrusion molding or blow molding. The polypropylene can benucleated or non-nucleated and it can contain heat and light Fstabilizers, antistatic agents, antioxidant, nanoparticles as well asfillers, such as mica, calcium carbonate, talc and wollastonite, andpigments, such as carbonate, titanium dioxide, carbon black,quinacridone, phtalocyanine blue and isoindolinone. Preferably, thepigment is either titanium dioxide, making the material white, or carbonblack, making the material black. More preferably, the pigment istitanium dioxide. Other thermoplastic resins suitable for the presentpurposes are various high-quality polyethylene, polybutylene andpoly(ethyelene terephthalate) grades.

Preferably, either the tube or the cap is fabricated from athermoplastic material containing a colorant, such as a pigment,nanoparticles or another agent altering the light transmittingproperties of the material, while the other is fabricated from athermoplastic material lacking such a colorant. More preferably, thematerial of the tube contains colorant, while the material of the caplacks colorant. Most preferably, the thermoplastic material ispolypropylene and the colorant is chosen from agents, which make thematerial opaque.

The thickness of the tube wall is preferably about 0.002 inches to about0.030 inches (approximately 0.05 mm to 0.76 mm), more preferably 0.002inches to about 0.0065 inches (approximately 0.05 mm to 0.17 mm) or evenmore preferably 0.002 inches to about 0.009 inches (approximately 0.05mm to 0.23 mm), the achievable thickness being dependent upon size ofarea and part geometry.

Particularly, the wall thickness of the upper wall portion 14 can be,for example 0.009-0.030 inches (0.23 mm to 0.76 mm). The lower wallportion 16 can be manufactured to have a uniform wall thickness of0.0025 to 0.0065 inches (approximately 0.06-0.17 mm). The consistency ofthe thickness is high with the maximum deviation from the desired wallthickness usually being below 25%, even below 10%, depending on theshape of the tube and the desired wall thickness. This leads to an evenheat transfer to the reagent sample. That is, the thermal contributionof the vessel diminishes as its mass becomes smaller in relation to themass of the sample.

The thickness of the dome-shaped or flat portion 9 of the cap can be,for example 0.002-0.009 inches (0.05-0.23 mm), whereas the thickness ofthe cylindrical portion 8 of the cap can be, for example 0.002-0.030inches (0.05-0.76 mm).

The tube and the cap, as mentioned above, comprise a one-piece assembly.The portions, i.e. the tube and the cap, of the assembly are preferablyconnected by way of a hinge strap or device, herein also called ahinge-like structure. The material of this hinge-like structure ispreferably polypropylene with or without colorant.

According to a preferred embodiment of the present invention, theabove-mentioned assembly comprises a single tube attached to a singlecap through a hinge-like structure. The tube may have any conventionaltube size, such as 0.2 ml, 0.5 ml, 0.6 ml, 1.0 ml, 1.5 ml or 2.0 ml. Foruse in PCR the tube is preferably dimensioned to fit 0.2 ml or 0.5 ml ofliquid.

According to another preferred embodiment of the present invention, theabove-mentioned tube-and-cap assembly comprises a strip of tubes,wherein each tube is attached to a single cap through a hinge-likestructure. One strip preferably comprises 8 or 12 tubes. The tubes inthe strip of tubes may have any conventional tube size, such as 0.2 ml,0.5 ml, 0.6 ml, 1.0 ml, 1.5 ml or 2.0 ml.

According to another preferred embodiment of the present invention, theabove-mentioned tube-and-cap assembly comprises a strip of tubesattached to a strip of caps through one or more, preferably one or two,more preferably only one, hinge-like structure. As mentioned above, onestrip of tubes preferably comprises 8 or 12 tubes. Further, one strip ofcaps also preferably comprises 8 or 12 caps. The tubes in the strip oftubes may have any conventional tube size, such as 0.2 ml, 0.5 ml, 0.6ml, 1.0 ml, 1.5 ml or 2.0 ml.

The sample vessel assembly of the present invention will be compatiblewith general laboratory equipment and analytical instrumentation thatare designed to accept tubes or strips of tubes of the above-mentionedsizes. Such general lab equipment includes centrifuges, thermal cyclers,simple heaters and chillers and liquid handlers, and the analyticalinstrumentation includes DNA automated sequencing systems, emission andcalorimetric plate readers, and PCR instruments, such as real-time,quantitative PCR instruments. The tubes should be capable of placementinto unrestricted heat transfer connection with the holder/heating meansof the analyzing equipment. Preferably, the sample vessel assembly isfabricated using materials making it suitable for optical or visualdetection.

In a typical application, the sample vessel assembly of the presentinvention is used for performing a PCR process in a thermal cycler. Suchcyclers comprise a sample holder, which is designed to receive the tubeor the strip of tubes and to provide a thermal pathway between aheating/cooling element of the device and the sample vessels.Preferably, the assembly is used for performing a qPCR process, morepreferably for performing a qPCR process using the SYBR green method orthe fluorescent reporter probe method, most preferably using thefluorescent reporter probe method. Other typical applications for theassembly include centrifuging, heating, chilling, storing, sequencingand other analytical applications.

According to a preferred embodiment of the present invention, the vesselhaving an integrated tube and cap is fabricated by use of a two-stepmolding process.

A “two-step molding process”, as used in the present invention, refersto a molding technique, wherein the high pressure injection moldingmachine and the specific mold can accommodate the introduction of twoseparate resins in one molding cycle which consists of two polymerinjection steps, one for each mentioned color, e.g. an opaque color andan optically clear color, a reflective color and an optically clearcolor, or white and black. Common plastics molding techniques includeinjection molding, pressure forming, vacuum forming, extrusion moldingor blow molding. When manufacturing bi-component or multi-componentplastic articles, such as in some cases when manufacturing toothbrushes,a two-step or multi-step molding technique can be used. Whenmanufacturing the vessels of the present invention, it is preferred touse a two-step injection molding technique, as described above.

In injection molding, the material, such as the plastic, to be moldedgenerally is added to the molding device in the form of pellets. Ifanother material, such as a colorant, is to be added, it is done in theinjection stage. Particularly when manufacturing thin-walled structures,the color of the molded structure may remain inhomogeneous, due to theuneven distribution of the colorant. In the present invention, toresolve this problem the plastic material and the colorant are firstpre-mixed, whereby the pellets added to the molding device alreadycontain an essentially homogeneous mixture of plastic and colorant.

In the two-step manufacturing process of the present invention, thefirst step is the molding of either the tube or cap component and thesecond step is the molding of the complementary component, e.g. cap withtube or vice versa, allowing the joining of the two different pigmentsin the area of the hinge strap.

The present invention also provides a method for achieving an accurateoptical signal in the detection of a thermal cycling process, whereinthe sample to be detected is located in a vessel of the presentinvention and the signal is obtained by sending excitation light throughthe cap of the mentioned vessel and detecting the emitted signalreturning through the same mentioned cap of the mentioned vessel.

According to a preferred embodiment of the invention, tube-to-tube lighttransmission is prevented in the above described method by the presenceof a colorant comprised in the tube material.

1. A vessel comprising an integrated tube-and-cap assembly, wherein oneor more tube(s) is connected by way of one or more hinge strap(s) ordevice(s) to one or more cap(s), characterized in that the assembly hasbeen fabricated in one piece, the tube and the cap have different lighttransmitting properties, since the material of either one or both of thetube and the cap comprises one or more colorants, which alter its lighttransmitting properties, and the materials of the tube and the cap donot comprise the same colorant or mixture of colorants.
 2. The vessel ofclaim 1, wherein the material of the tube comprises one or morecolorants and the material of the cap lacks colorant.
 3. The vessel ofclaim 1, wherein the material of the cap comprises one or more colorantsand the material of the tube lacks colorant.
 4. The vessel of claim 1,wherein the material of the tube comprises one or more colorants and thematerial of the cap comprises a different colorant or different mixtureof colorants than the material of the tube.
 5. The vessel of any ofclaims 1 to 4, wherein the cap is fabricated from a homogenous,optically clear material.
 6. The vessel of claims 5, wherein theoptically clear material comprises polypropylene.
 7. The vessel of claim1, wherein the tube is fabricated from an opaque material.
 8. The vesselof claim 7, wherein the opaque material comprises a homogenous mixtureof polypropylene and one or more colorants.
 9. The vessel of claim 1,wherein the colorants are chosen from pigments, nanoparticles or otheragents capable of altering the light transmitting properties of amaterial.
 10. The vessel of claim 9, wherein the colorant is titaniumdioxide, giving the material a white color, or carbon black, giving thematerial a black color.
 11. The vessel of claim 10, wherein the colorantis titanium dioxide.
 12. The vessel of claim 10, wherein the colorant iscarbon black.
 13. The vessel of claim 1, wherein the integratedtube-and-cap assembly comprises a single tube connected to a single capby way of a hinge strap or device.
 14. The vessel of claim 1, whereinthe integrated tube-and-cap assembly comprises a strip of tubes, whereineach tube is connected to a single cap by way of a hinge strap ordevice.
 15. The vessel of claim 1, wherein the integrated tube-and-capassembly comprises a strip of tubes, wherein the tube strip is connectedto a strip of caps by way of one or more hinge straps or devices. 16.Use of a vessel of claim 1 for thermal cycling, centrifuging, heating,chilling, storing, sequencing and other analytical applications.
 17. Useof a vessel of claim 1 for performing a PCR process.
 18. A method forachieving an accurate optical signal in the detection of a thermalcycling process, wherein the sample to be detected is located in avessel of claim 1 and the signal is obtained by sending excitation lightthrough the cap of the mentioned vessel and detecting the emitted signalreturning through the same mentioned cap of the mentioned vessel.